rhBMP_2对人牙髓干细胞分化及Delta蛋白表达的影响

Effects of rhBMP_2 on differentiation of human dental pulp stem cells and expression of Delta protein in vitro

目的:研究重组人骨形成蛋白2(rhBMP2)对人牙髓干细胞分化及Delta蛋白表达的影响,为人牙髓干细胞的研究提供理论依据。方法:酶消化法获得人牙髓干细胞,光镜下进行形态学观察,免疫荧光法检测细胞表面标志的表达;取第5代牙髓干细胞,分为实验组(加含50μg·L-1 rhBMP2的培养液)及阴性对照组(只加入培养液),检测碱性磷酸酶活性和Delta蛋白表达的变化。结果:人牙髓干细胞呈集落状生长,免疫组织化学检测显示nestin、vimentin染色呈阳性,免疫荧光法检测STRO-1呈阳性。与阴性对照组比较,实验组第7、14及21天碱性磷酸酶活性均明显升高(P<0.01),第21天Western blotting法检测Delta蛋白表达明显升高(P<0.05)。结论:培养的牙髓干细胞具有干细胞特性,并且rhBMP2可使其碱性磷酸酶活性增高并上调Delta蛋白表达,提示Delta蛋白可能参与牙髓干细胞向成牙本质样细胞分化的过程。

Objective To investigate the effects of rhBMP2 on the differentiation of human dental pulp stem cells and expression of Delta protein in vitro, provide a theoretical basis for research on human dental pulp stem cells. Methods Monocell suspension was separated from the human adult dental pulp with collagenase I and dispase digestion. Clonogenic cells were observed under light microscope and the expressions of surface markers were determined with immunofluorescence, divided into experimental group （containing 50μg·L^-1 rhBMP2 in the culture medium） and negative control group （containing culture medium only）, alkaline phosphatase and the expression of Delta proterin of the ceils at at passage 5 were detected. Results The human dental pulp stem ceils showed colony growth, the nestin and vimentin staining were positive by immunohistochemical staining. STRO-1 was positive in cells by immunofluorescence. Compared with negative control group, the activities of alkaline phosphatase increased after treatment with rhBMP2 for 7, 14 and 21d （P〈0.01）, the expression of Delta protein was higher after treatment with rhBMP2 for 21d by western blotting （P〈0.05）. Oonclusion The cultured human dental pulp stem cells have have the characteristics of stern cells, rhBMP2 may increase the activity of alkaline phosphatase and up- regulate the expression of. Delta protein may be involved in the differentiation process of Human dental pulp stem cells in to odontoblasts like cells.